Lymphocytes are the main cells of the immune system responsible for acquired immunity and the immunologic characteristics of diversity, specificity, memory and self/nonself recognition. Lymphocytes can be broadly divided into B cells, characterized by the presence of membrane-bound immunoglobulin (antibody) molecules which serve as receptors for, and can bind, soluble antigens; and T cells, characterized by the presence of membrane-bound receptor molecules (TCR, for T-cell receptor), which recognize and bind antigen only when the antigen is associated with a self-molecule encoded by genes within the major histocompatibility complex (MHC). The MHC is referred to as the H-2 complex in mice and as the HLA (for human leukocyte antigen) complex in humans.
T-cell recognition of antigen is the basis of the adaptive immune response, which is the ability of the immune system to selectively recognize, neutralize and obliterate infectious pathogens or pathological cells. Effector T cells generated in response to antigen are responsible for cell-mediated immunity. All T-cell subpopulations express the TCR, but they can be distinguished by the presence of one or the other of two membrane glycoprotein molecules on their surfaces (surface antigens), CD4 or CD8, and their role in the immune response. Thus, two major subpopulations of T cells can be characterized: the CD4+ or helper T cells (TH), which facilitate the activities of other cell types, and the CD8+ or cytotoxic T lymphocytes (CTL), which can directly kill abnormal or infected cells.
CD4+ T cells are further divided into TH1 inflammatory T cells, which secrete various cytokines (TH1 response) that activate mainly T cytotoxic cells and macrophages responsible for the intracellular destruction of phagocytosed microorganisms, and TH2 T cells, which secrete various cytokines (TH2 response) that activate B cells to produce antibodies. CD8+ T cells generally function as CTLs. Another T-cell related lymphocyte subset is the natural killer cells (NK), which are large, granulated lymphocytes displaying cytotoxic activity against a wide variety of tumor and other abnormal cells.
T-cell recognition is mediated by direct interaction of the TCR with an antigenic peptide displayed by a MHC product on an antigen-presenting cell (APC). As a result of specific interaction between TCR of a T lymphocyte that has not yet interacted with antigen (naive or unprimed cell) with MHC molecules displaying antigenic peptides on a professional APC (e.g. dendritic cells, macrophages, B cells), the T cell is activated and it proliferates and eventually differentiates to produce effector cells, which function as TH cells or CTLs to eliminate the antigen, and memory cells, which are responsible for the life-long immunity observed for many pathogens.
In mammals, there are two types of MHC molecules: MHC class I molecules, which are present on almost all nucleated cells in the body and are recognized by CD8+ cells, and MHC class II molecules, which are mainly expressed on professional APCs of the immune system and are recognized by CD4+ cells. Thus, CD8+ T cells, which generally function as CTLs, are said to be class I restricted, and CD4+ T cells, which generally function as TH cells, are said to be class II restricted.
Class I MHC molecules are composed of a polymorphic heavy chain (α) non-covalently associated with a monomorphic (in humans) non-MHC encoded light chain protein of about 12 kDa, termed β2 microglobulin (β2m). The heavy α chain is a polymorphic transmembrane glycoprotein of about 45 kDa consisting of 3 extracellular domains, each containing about 90 amino acids (α1 at the N-terminus, α2 and α3), a transmembrane region of about 40 amino acids and a cytoplasmic tail of about 30 amino acids. The α1 and α2 domains, the membrane distal domains, form the peptide-binding groove or cleft having a sufficient size to bind a peptide of 8-10 amino acids, whereas the α3 domain is proximal to the plasma membrane. β2m has a single immunoglobulin (Ig)-like domain, not anchored to the plasma membrane, and interacts mainly with the α3 chain, which also possesses a characteristic Ig fold. In humans, there are three α chain genes, called HLA-A, HLA-B and HLA-C, for each of which multiple alleles have been identified. In mice, there are three α chain genes, called H-2K, H-2D and H-2L. CD8+ T cells recognize peptides in the context of MHC class I molecules.
Class II MHC molecules contain two different polypeptide chains, a 33-kD α chain and a 28-kDa β chain, which associate by noncovalent interactions. Like class I MHC molecules, class II MHC molecules are membrane-bound glycoproteins that contain extracellular domains, a transmembrane segment and a cytoplasmic tail. Each chain in these noncovalent heterodimeric complexes contains two extracellular domains: α1 and α2 domains and β1 and β2 domains. The membrane-distal domain of a class II molecule is composed of the α1 and β1 domains and forms the peptide-binding groove or cleft having a sufficient size to bind a peptide, which is typically of 13-18 amino acids. The membrane-proximal domains, α2 and β2, have structural similarities to Ig constant (C) domains. Three pairs of class II α and β chain genes exist in humans, known as HLA-DR, HLA-DP and HLA-DQ. The highest level of polymorphism is documented for HLA-DR. This polymorphism is solely contributed by the DRβ chain, as DRα is monomorphic. In mice, the pairs of genes are called H-2IA and H-2IE. CD4+ TH cells recognize peptides in the context of class II MHC molecules.
All T cells bind their specific MHC::peptide antigen via clone-specific or clonotypic TCR molecules. TCRs are disulfide-linked heterodimeric transmembrane proteins made of α and β chains. The N-terminal variable (V) domains of these chains, which together form the antigen-binding site, are similar to those of Ig variable (V) chains, whereas the membrane-anchored C-terminal domains are analogous to Ig constant (C) domains.
On the T-cell membrane, the clonotypic TCR associates non-covalently with CD3, forming the TCR-CD3 membrane complex. CD3, the signal transduction element of the TCRs, is composed of a group of invariant proteins called gamma (γ), delta (δ), epsilon (ε), zeta (ζ) and eta (η) chains. The γ, δ and ε chains are structurally-related, each containing an Ig-like extracellular constant domain followed by a transmembrane region and a cytoplasmic domain of more than 40 amino acids. The ζ and η chains have a distinctly different structure: both have a very short extracellular region of only 9 amino acids, a transmembrane region and a long cytoplasmic tail containing 113 and 115 amino acids in the ζ and η chains, respectively. The invariant protein chains in the CD3 complex associate to form noncovalent heterodimers of the ε chain with a γ chain (εγ) or with a δ chain (εδ) or of the ζ and η chain (ζη), or a disulfide-linked homodimer of two ζ chains (ζζ). About 90% of the CD3 complex incorporate the ζζ homodimer.
The cytoplasmic regions of the CD3 chains contain a motif designated the immunoreceptor tyrosine-based activation motif (ITAM). This motif is found in a number of other: receptors including the Ig-α/Ig-β heterodimer of the B-cell receptor complex and Fc receptors for IgE and IgG. The ITAM sites have been shown to associate with cytoplasmic tyrosine kinases and to participate in signal transduction following TCR-mediated triggering. In CD3, the γ, δ and ε chains each contain a single copy of ITAM, whereas the ζ and η chains harbour three ITAMs in their long cytoplasmic regions. Indeed, the ζ and η chains have been ascribed a major role in T cell activation signal transduction pathway (Howe and Weiss, 1995; Sherman and Chattopadhyay, 1993).
In mammals, T cell maturation occurs in the thymus. During maturation, a distinct mechanism operates to ensure positive selection, namely that αβ TCRs expressed in a given individual cell will recognize and bind to self-MHC molecules, as well as negative selection, namely to eliminate those T cells bearing high affinity TCRs which may interact with self-MHC molecules alone or self-antigens plus self-MHC molecules, that would pose the threat of an autoimmune response if they matured. Hence, the mature T-cell repertoire can provide an adequate defense against pathogens, while avoiding response against self-antigens. However, peripheral tissue antigens often fail to be adequately presented in the thymus, and potentially self-reactive T cells do succeed to exit the thymus, while downstream mechanisms, which induce peripheral tolerance are not always sufficient to inactivate these potentially harmful T cells. In addition, no mechanism has evolved to eliminate or inactivate T cells capable of reacting against foreign MHC allelic products, referred to as alloreactive cells. As a result, the action of harmful T cells may inflict severe damage and may be associated with life-threatening situations commonly associated with T-cell mediated diseases and conditions such as autoimmune diseases and transplantation, and with responses against innocuous foreign antigens, resulting in hypersensitivity reactions such as allergy and asthma.
Autoimmune disorders are characterized by reactivity of the immune system to an endogenous antigen, with consequent injury to tissues. More than 80 chronic autoimmune diseases have been characterized that affect virtually almost every organ system in the body. The most common autoimmune diseases are insulin-dependent diabetes mellitus (IDDM), multiple sclerosis (MS), systemic lupus erythematosus (SLE), rheumatoid arthritis, several forms of anemia (pernicious, aplastic, hemolytic), thyroiditis, and uveitis. Autoimmune disorders are far more prevalent in women and are among the top 10 causes of death of young and middle-aged women in the U.S.A.
Autoimmune diseases result from sustained adaptive immune responses mounted against innocuous self-antigens. The effector mechanisms that eventually cause tissue damage and disease are most likely those that take part in normal adaptive responses, and include production of specific antibodies, generation of immune complexes, inflammatory and cytotoxic T cells and activated macrophages.
The role of T cells in autoimmune diseases has been extensively studied in MS, a chronic inflammatory disease of the central nervous system (CNS) and its rodent model experimental autoimmune encephalomyelitis (EAE), (for review, see Hafler and Weiner, 1995). In MS, activated CD4+ T cells found in the central nervous system (CNS) display specificity to a number of abundant CNS proteins, including myelin basic protein (MBP), proteolipid protein (PLP), myelin oligodendrocyte-associated protein (MOG) and S-100. Susceptibility to MS is associated with the HLA-DR2 haplotype approximately 50-70% of MS patients carry the DR2 allele, which is found in only 20-30% of normal individuals. This association has enabled, for example, the identification of immunodominant MBP peptides using panels of HLA-DR2-restricted T cell clones (Wucherpfennig, et al., 1991).
A large bulk of data has also been accumulated in IDDM along with its non-obese diabetic (NOD) mouse model. In IDDM, CD4+ T cell autoantigens include insulin, GAD (glutamic acid decarboxylase) 65, GAD67, hsp (heat-shock protein) 65, and ICA (islet-cell antigen) 69 (Roep, 1996). Recently, a study of the NOD mouse system, which employed a novel screening strategy, has enabled the identification of an insulin B-chain peptide as the first CD8+ T cell epitope in an autoimmune disease (Wong, et al., 1999).
In summary, a limited number of peptides derived from proteins involved in autoimmune diseases are associated with the onset of the disease. The immune responses to self-antigens are maintained by the persistent activation of self-reactive T cells. Removal of T cell populations that are associated with the autoimmune response should lead to prevention and/or cure of the disease. This model was demonstrated in the NOD mice, where the removal of T-cell populations that recognize proinsulin II, prevented the onset of IDDM (French, et al., 1997).
Allograft rejection typically results from an overwhelming adaptive immune response against foreign organ or tissue. It is the major risk factor in organ transplantation and is the cause of post-transplantation complications. A major complication associated with bone marrow (BM) transplantation, known as graft-versus-host (GVH) reaction or graft-versus-host disease (GVHD), occurs in at least half of patients when grafted donor lymphocytes, injected into an allogeneic recipient whose immune system is compromised, begin to attack the host tissue, and the host's compromised state prevents an immune response against the graft Alloreactivity is complex and involves many cell types as well as inflammatory factors. It is largely mediated by both CD8+ (CTL) and CD4+ (TH) T cells (for review, see Douillard et al., 1999; Hernandez-Fuentes et al., 1999; Pattison and Krensky, 1997).
Allograft rejection results from proper recognition of foreign MHC and activation of the adaptive immune system and is carried out by direct or indirect pathways. The direct pathway, where T-cell receptors directly recognize intact allo-MHC with or without bound peptides on the surface of target cells, apparently accounts for most of the CTL function. The indirect pathway, where T-cell receptors recognize MHC allopeptides after processing and presentation, leads to the activation of T helper cells. These cells provide the necessary signals for the growth and maturation of effector CTLs and B cells leading to rejection (Sherman and Chattopadhyay, 1993; Watschinger, 1995).
The actual role of specific peptides in direct allorecognition is ambiguous. Some studies demonstrate that allorecognition is peptide-independent (Mullbacher et al., 1999; Smith et al., 1997), while others imply that specific peptides do contribute to allorecognition (Wang, et al., 1998). Allorecognition may, therefore, comprise peptide-independent, peptide-dependent or peptide-specific interactions.
Ideally, treatment of autoimmune diseases should reduce only the autoimmune response while leaving the rest of the immune system intact. In the absence of such treatments, current therapies of autoimmune diseases include non-immune-specific treatments via a broad spectrum of immunosuppressive drugs such as corticosteroids, cyclosporine A, methotrexate and tacrolimus (FK506). However, these agents have severe side effects such as “generalized immunosuppression” and toxicity.
Successful transplant engraftment and effective treatment of autoimmune diseases would be greatly facilitated by tolerizing, inactivating or eliminating harmful, or potentially harmful, T-cells. In both cases, specificity (or at least high selectivity) of the therapy is mandatory if severe side effects are to be minimized. For maximal efficacy and broad applicability, anti-T cell protocols are to target the whole repertoire of (allo or auto)specific T cell clones, and to act independently of HLA identity. Indeed, design and evaluation of specific treatment strategies have been the objective of numerous studies in both these fields. Among the approaches that were explored, are anti-T cell idiotypic manipulation (Bluestone et al., 1986), reduction of antigen presentation by the graft (Carpenter et al., 1976; Faustman, 1995) and suppression of T cell precursors by veto cells (Miller et al., 1988; Reich-Zeliger et al., 2000; Thomas et al., 1991).
Identification of potential autoantigens and characterization of autoantigen-specific T cell clones, mainly in MS (or EAE) and IDDM, have prompted active research aimed at developing specific immunotherapies to autoimmune diseases. Experimental therapeutic approaches include T-cell vaccination with autoreactive T cells (Ben-Nun and Cohen, 1981) or their TCR peptides (Howell et al., 1989; Vandenbark et al. 1991), tolerance induction by oral antigens (reviewed in Hafler and Weiner, 1995), peptide blockade of MHC molecules by specific peptides associated with the disease or their analogues (Elias et al., 1991; Windhagen et al., 1995), and monoclonal antibody treatment However, wide clinical applicability of these approaches still has to be demonstrated. Undoubtedly, novel solutions are to be constantly pursued so that accumulating knowledge and understanding of the immune system are eventually reduced to wide medical practice.
Specific direction of an immune intervention procedure against the pool of auto-or allo-specific T-cell clones demands a well-defined structural or functional common denominator which can serve as an identification tag for this pool. For example, the concept of vaccination with TCR peptides or whole autoreactive T cells (Howell, et al., 1989; Vandenbark, et al., 1991) is based on the observation that encephalitogenic T cells in EAE utilize only a restricted set of germ-line TCR Vα and Vβ genes, hence, their TCR amino acid sequences constitute a potential (although not fully specific) marker. However, autoreactive T cell clones isolated in MS are more promiscuous in their TCR usage and are thus less accessible to this strategy. The trivial common denominator of the whole panel of T cell clones recognizing a given MHC::peptide ligand is the ligand itself, irrelevant of TCR gene products of those clones. The problem is how this understanding can be exploited so that those cells can be specifically targeted for therapy. A novel mechanism, devised to trigger a potent reaction against those T cells following their specific interaction with the ligand is a likely solution. This can only be achieved if the ligand is engineered to be linked to an adequate effector function, activated upon its engagement with specific TCRs.
In recent years, the inventors (Eshhar et al., 1993; Gross et al., 1995; Gross et al., 1989; Hwu, et al., 1993) and others (reviewed in Gross and Eshhar, 1992), have demonstrated that genetic engineering enables redirecting T cell recognition at will via chimeric activation receptors. This was accomplished by replacement of the ligand-binding domain of a T-cell receptor, with binding domains derived from other receptor molecules. Other studies have shown that reciprocal substitution of transmembrane and intracellular domains of surface receptors with those of different molecules involved in T-cell activation signal transduction leads to a corresponding change in the pattern of response to the same signal. The transmembrane and cytoplasmic regions of the CD3 ζ chain or Ig Fc receptor γ chain have been frequently used in such experiments, proving most powerful in this regard (e,g. Eshhar et al., 1993; Irving and Weiss, 1991; Letourneur and Klausner, 1991; Romeo and Seed, 1991).
A number of publications disclose chimeric receptors comprising a CD3 ζ chain and an extracellular binding domain. For example, Seed et al., in U.S. Pat. No. 5,843,728, disclose chimeric receptors comprising the extracellular domain of CD4 fused to an intracellular portion of a TCR CD3 ζ or η chain, a B-cell receptor polypeptide or an Fc receptor polypeptide. T lymphocytes expressing these chimeras would recognize and kill cells expressing HIV gp120.
U.S. Pat. No. 5,855,740 and U.S. Pat. No. 5,834,266 disclose chimeras comprising an intracellular CD3 ζ chain and an extracellular domain capable of specifically binding to at least one ligand. U.S. Pat. No. 5,359,046 discloses chimeras optionally containing an intracellular CD3 ζ chain fused to extracellular domains derived from CD4, CD8, Ig or single-chain antibody. U.S. Pat. No. 5,712,149 discloses chimeric costimulatory receptors whose intracellular domain is derived from CD2 or CD28 and may, in addition, comprise a CD3 ζ chain domain.